The overall purpose of the proposed studies is to elucidate the role of the membrane mucin Muc4 in promoting breast tumor initiation and progression. Muc4 harbors potent anti-adhesive and growth signaling activities when expressed in cultured cells, which in principle could play roles in both the initial transformation of healthy epithelia to early hyperplastic phenotypes and in later transitions to highly invasive and metastatic states. Consistent with this, it has been reported that Muc4 is overexpressed and mislocalized in up to 20% of local breast tumors compared to patient-matched normal tissue samples, and up to 40% of lymph node metastases relative to matched primary tumor. This project will integrate these observations by providing an analysis of Muc4 contribution to the development of progressively more aggressive states in vitro and in vivo. The driving hypothesis is that the aberrant expression of Muc4 protein in breast tumors facilitates their progression to malignancy. Aim 1 will analyze the outcome of inducible Muc4 overexpression in a series of isogenic cultured breast epithelial cell lines derived from MCF10A cells that model normal, hyperplastic, and malignant breast tissue, as well as in breast tumor cell lines that model ErbB2-positive, ER-positive and triple-negative disease. Analysis of cellular growth properties and signaling pathway usage will be carried out when cells are grown in 2D and 3D culture. Comparison of full-length Muc4 with a tumor-associated splice variant lacking its VNTR domain will reveal the role of Muc4 anti-adhesive activity in regulating cellular growth properties. In Aim 2, Muc4 will be inducibly expressed in the mammary glands of transgenic mice, and these will be crossed into mouse models of breast cancer. Induction of Muc4 expression at two different points in tumor development will permit the in vivo assessment of Muc4 contribution to tumor progression at different stages of disease. The impact of Muc4 knockout on mouse mammary tumor development and progression will also be analyzed. Together the studies of Aims 1 and 2 will determine whether the Muc4 overexpression observed in patient tumors contributes to breast cancer progression. Aim 3 will examine the mechanisms of Muc4 dysregulation in patient samples, and will begin to assess the role of a novel endoplasmic reticulum-localized, TGF-regulated protein degradation pathway in governing cellular Muc4 levels. Overall, these studies will validate Muc4 as a novel contributor to breast cancer progression, and will begin to unravel biochemical pathways that might ultimately be exploited for therapeutic intervention.